Detailed computer-assisted comparative analysis of protein sequences & gene arrangement in positive-strand, negative-strand and double-stranded RNA viruses was performed in order to predict functions of gene products, to deriveprinciples of genome organization, and to propose evolutionary scenarios. RNA viruses have small genomes and complete genome sequences representative of the majority of known groups of these viruses are currently available allowing for large-scale comparative and evolutionary studies. Positive-strand RNA viruses have a limited number of conserved proteins that are mostly involved in genome replication and expression. Two types of putative methyltransferase domains, one of which is conserved in viruses of the alpha-like supergroup, and the other in flaviviruses, have been identified. Domain organization of Hepatitis E virus polyprotein was explored in detail and it was shown that this virus is related to Rubella virus and to Beet necrotic yellow vein virus, a plant furovirus. Evolution of these viruses appeared to involve several genome rearrangement events. Putative papain-like protease domains were identified in the polyproteins of each of these viruses. Analysis of the genome of Beet yellows closterovirus revealed a unique gene organization, with genes coding for proteins related to the respective proteins of two distinct groups of positive-strand RNA viruses, and to cellular heat shock proteins. It was shown that the coat protein gene of this virus is duplicated. This is the first example of coat protein gene duplication in viruses with elongated particles. A general concept of evolution of positive-strand RNA viruses based on the interplay between conservation of gene arrays and gene module shuffling is being developed. Work on summation of the information on virus genome organization and derivation of evolutionary schemes is in progress. Comparison of the sequences of proteins encoded by double-stranded RNA showed very limited conservation. The only sequence conserved among different groups was that of a domain of the RNA-dependent RNA polymerase. This sequence is distantly related to the polymerases of positive-strand RNA viruses. Phylogenetic analysis suggested that double-stranded RNA viruses may not be a monophyletic group. Different groups of double-stranded RNA viruses could have evolved from distinct groups of positive-strand RNA viruses. The significance of the project lies in the unique opportunity to compare the organization of a large number of complete genomes with varying degree of similarity to each other and to gain insight into the possible pathways of evolution of the genetic diversity of vast classes of viruses.